The present invention relates to an open tubular capillary column preferably usable for high-speed micro liquid chromatography and the manufacturing method therefor, more particularly, to the provision of an open tubular capillary column capable of high sensitive detection and microanalysis, having a stable stationary phase.
In recent years, liquid chromatography has made a rapid progress along with, or with the aid of, the development of high-pressure pumps or the enhancement of separating capability of column packing materials. As a high-speed system, wherein a mobile phase is flowed in high-speed for rapid separation, liquid chromatography has been recognized to be a highly effective separating and analytical technique in the field of chemistry, biology, medical science, etc.
Columns used for such a high-speed liquid chromatography are required, from the standpoint of increasing of the separating speed, high capability of microanalysis, high degree separating capability, economy of column packing materials or media, etc., to be as small as possible in the inner diameter thereof. In the actual practice, columns of 2.5 mm, more or less, are widely used. Further diminishing of the inner diameter down to 1.0 mm to 0.5 mm has been conventionally deemed very difficult or impracticable, because small recesses, jogs, or roughness on the inner wall surface of the column may directly affect the separating capability.
A column of the inner diameter approximately 0.5 mm and the relevant technology has been disclosed in the Japanese Patent Application TOKU-GAN-SHO-49 (1974)-72107 as well as in the U.S. Pat. No. 4,059,523. This column may be used in a high-speed liquid chromatography at the flow rate of 10 .mu./min. more or less. Even such a small diametered column of glass tube, however, is not free from using a column packing material; and packing a column packing material to the column with such a small diameter is not an easy job.
In the gas chromatography, on the other hand, a so-called Golay column, a kind of open tubular capillary column in which no column packing is used but a liquid phase (stationary phase) is instead held, is known. This Golay column enables one to eliminate the troublesome packing operation of the column packing material. It is quite natural therefore that this art led the researchers in the field to the application of an open tubular capillary column of this type even to liquid chromatography. No successful cases, however, have been reported so far. This may be attributable to a fundamental difference of the mobile phase between gas chromatography and liquid chromatography. Before applying the art in gas chromatography to that in liquid chromatography, difference in the capillary column design due to the difference of the diffusion coefficient should be solved first. This is the very problem that motivated this invention. This is the very reason that the technology in gas chromatography can not be applied as it is to liquid chromatography.
We thought the essential problem for obtaining an ideal column for liquid chromatography is to determine the design conditions for relatively diminishing the diffusion of the sample in the mobile phase liquid. We found, after a series of experiments, that the inner diameter of the column of glass tube should be 0.10 mm or less and the length thereof should be determined as to make the net inside capacity 0.5 .mu.l or more, preferably 1.0 .mu.l or more. A column satisfying such conditions have been found to be of very high separating capacity at a high theoretical plate number. Another problem must be solved before practically applying such a column to the liquid chromatography, i.e., the problem of coating a stationary phase on the inner surface of the column of glass tube, which is a prerequisite condition therefor. A proper measure must be taken for preventing solution of the stationary phase into the mobile phase, as well as the accompanying exfoliation or mechanical scaling-off of the stationary phase, because the stationary phase is soluble into the mobile phase, although slightly, and the mobile phase runs fast when a high-speed separation is executed. Further research for preventing the mechanical scaling-off of the mobile phase in such a small diametered thin column has led us to the under-mentioned discovery. Noticing that glass, which constitutes a principal material of the column, has on its surface silanol groups, we thought of chemically bonding this kind silanol group with a silane or its derivative, which is to become a stationary phase, for forming a stable layer of silane or its derivative chemically bonded on the inner surface of the column of glass. It has been found that a layer of silane or any derivative thereof is very useful as a stationary phase. This invention has thus been completed on such a basic idea.